Portland State University Portland State University

PDXScholar PDXScholar

Dissertations and Theses Dissertations and Theses

1984

Effects of dehydration on hemoglobin oxygen affinity Effects of dehydration on hemoglobin oxygen affinity

and blood cell volume in two anurans and blood cell volume in two anurans

Andrew Christopher Zygmunt Portland State University

Follow this and additional works at: https://pdxscholar.library.pdx.edu/open_access_etds

Part of the Biology Commons, and the Physiology Commons

Let us know how access to this document benefits you.

Recommended Citation Recommended Citation Zygmunt, Andrew Christopher, "Effects of dehydration on hemoglobin oxygen affinity and blood cell volume in two anurans" (1984). Dissertations and Theses. Paper 3424. https://doi.org/10.15760/etd.5304

This Thesis is brought to you for free and open access. It has been accepted for inclusion in Dissertations and Theses by an authorized administrator of PDXScholar. Please contact us if we can make this document more accessible: pdxscholar@pdx.edu.

AN ABSTRACT OF THE THESIS OF Andrew Christopher Zygmunt for

the Masters of Science In Blology presented August 3, 1984.

Title: Effects of Dehydration on Hemoglobin Oxygen Aff lnlty

and Red Blood Cel I Volume In Two Anurans.

APPROVED BY MEMBERS OF THE THESIS COMMITTEE:

Richard R. Petersen

The degree of terrestrial Ism In anurans Is correlated

with a differential tolerance to desiccation.

Cardiovascular insuff lclency and reduced oxygen del Ivery to

the tissues may be the mechanism of dehydratfonal death f n

amphibians.

Two aspects of posslble adaptation In cardlovascular

performance caused by Increased plasma electrolytes were

examined. Cel Is In anfsotonfc plasma may either act as

osmometers or volume regulate. Blood flow rate Is

dependent upon eel I viscosity, which Jn turn Is a

consequence of eel I volume and membrane deformabl I Jty.

Cel I volume changes which Increase membrane deformabll lty

wll I thus potentially extend the I lmlts of dehydration

tolerance. It was found In.&... catesbelana and~ marlnus

that red blood eel Is maintain constant volume during

2

dehydration. Cel Is In vitro Initially lose water, but then

sodium, potassium and water move Into the eel I. Cel I

viscosity within the physiologic range of hematocrlts was

higher In salt loaded non-regulating eel Is of~ marl nus

than In regulatlng Isotonic eel Is.

A consequence of water loss In non-regulating eel Is,

or uptake of Ions In regulating eel Is, Is an Increase of

lntracel lular Ion concentration. Hyperosmolaltty Influences

oxygen loading characteristics of blood. Ionic

Interactions are known mediators of hemoglobln function.

It was found In B.a.rut and .B.u.f.Q that Increasing lntracel lular

Ionic concentration falled to Influence the oxygen

dissociation curve. Adaptation was therefore not made to

Increase oxygen del lvered to the tissues during a time of

general clrculatory lnsuff lclency.

EFFECTS OF DEHYDRATION ON HEMOGLOBIN

OXYGEN AFFINITY AND BLOOD CELL

VOLUME IN TWO ANURANS

by

ANDREW CHRISTOPHER ZYGMUNT

A thesis submitted In partlal fulfll lment of the requirements for the degree of

MASTER OF SCIENCE f n

BIOLOGY

Portland State University

1984

TO THE OFFICE OF GRADUATE STUDIES AND RESEARCH:

The members of the committee approve the thesis of

Andrew Christopher Zygmunt presented August 3, 1984.

Richard S. Petersen

Department of Biology

Dean of Graduate Studies and Research

ACKNOWLEDGEMENTS

I want to thank Dr. Stan Hll Iman for Introducing me

to physlology and for sharing his tequlla. I have valued

the assistance and humor of Ors. Phi I Ip Withers, Richard

Petersen, and Larry Crawshaw; as wel I as that of my tel low

graduate students. Most Importantly, without the love and

caring of Diane this work would have been lmposslble.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS

LIST OF TABLES •

LIST OF FIGURES.

INTRODUCTION •

MATERIALS AND METHODS.

Anlmals •

Mean Cel I

•

Volume.

•

•

•

Hemoglobin Oxygen Aff lnlty.

lntracel lular Sodium and Potassium.

Viscosity

RESULTS. •

Mean Cel I Volume. • •

Hemoglobln Oxygen Affinity.

lntracel lular Sodium and Potassium.

Viscosity.

DISCUSSION

REFERENCES

• • • • • •

PAGE

11 I

v

vi

• •

8

8

8

1 1

12

• 13

• 13

13

15

15

15

26

• 32

TABLE

LIST OF TABLES

PAGE

lntracel lular sodium and potassium

concentrations CmEq/Kg dry mass) In the

erythrocytes of .IL. marlnys Cn=9) and B.... catesbelana

Cn=9). Values are means± standard error assuming

10% trapped plasma. Asterisk Indicates slgnlf lcant

difference Cp<0.025).......................... 17

LI ST OF FIGURES

FIGURE PAGE

1. In vivo volume of erythrocytes of .fL. marfnus

(water losses 28-36% lnltlal body mass)

and .B.a. catesbelana (water losses 14-20% lnltlal

body mass) •••• . . . . . . . . . . . . 2. Temp~rature dependence of RBC volume regulatfon

In .fL.. marlnus and .B... catesbelana. Rana,

18

CA9oc lsotonrc, e2ooc Isotonic, Agoc hypertonlc,

920oC hypertonfc). Ji.Llf.g, CY 9oC Isotonic,

•2ooc lsotonlc,~goc hypertonlcAt2ooc hypertonlc>

• • • • • • • • • • • • • • • • • • • • • • 1 9

3. Volume regulatlon of RBC's In .B... catesbelana

after 1.5 hours. Top I lne Indicates behavior as

osmometer, y=71.5 (±0.4)x-0.1 (±0.1), r=1.00.

Bottom lfne RBC's In normal Ringer's,

y=21.0 (±5.3)x-0.4 (±2.0), r=.78.

C • , potassium free media; Y , oubaln media;

• , normal Ringer's) ••• . . . . . . . . . . 20

vii

4. Volume regulatton of RBC's In .a... martnus after t.5

hours. Top I tne ts behavior asosmometer,

y=66.7 C±0.5)x+0.1 (±0.1), r=1.00. Bottom llne

RBC 1 s In normal Ringer's, y=26.9 C±2.8)x-0.3 C±t.t>

r=.94. CA , potassium free media; • , oubaln

media; • , normal Ringer's) •••••••••• 21

5. The relattonshlp between red eel I tntracel lular ton.

concentrations and plasma electrolytes In

.B... catesbelana corrected for 10% trapped plasma.

cesodtum y=0.5x-21, r=.75; A potassium y=1.1x+97,

r=.93.) • • • • • • • • • • • • • • • • • • • 22

6. The relatfonshtp between red eel I tntracel lular Ion

concentrations a~d plasma electrolytes In B.marlnus

corrected for .10% trapped plasma.

cesodtum y=0.5x-14, r=.73; Apotasslum y=0.4x+190,

r=.79) •••••••••••••••••••• 23

7. The relattonshtp between .a... marlnys hematocrlt and

the In transformation of red eel I viscosity at

a shear rate of 450/sec and 20oC. Sol Id I lne

Is salt loaded (180 mEq/L), In y=0.028C±.001)x+0.90

C±.04), r=.98. Dashed I fne ts Isotonic plasma,

In y=0.035 C±.002)x+0.43 C±.07), r=.93. • • • • 24

INTRODUCTION

Anurans are a diverse group of some 3000 species

which occupy habitats ranging from rain forests to deserts.

Although the vast majority of species are freshwater, their

number Includes species adapted for marine environments.

Modern anurans are descendants of the f lrst vertebrates to

face the desiccating environment of a terrestrlal habitat.

The examination of the phystologlcal correlates associated

with their degree of terrestrial Ism has been a fruitful

Inquiry.

Water balance In anurans has been described by a

number of workers. With a few exceptions, CPhyl tom~~,

Cblcomantls, and Hypecol lys), frogs and toads have

cutaneous evaporative water loss rates equal to a free

water surface (Bentley 1966a). Thorson (1955> found no

correlation between evaporative water loss and degree of

terrestrial ism, but did find a high correlatlon between

Increased tolerance to water loss and xerlc habitats.

Hll Iman (1980) found the aquatic species Xenopys laeyl~ to

tolerate 34% loss of lnltlal body mass, whereas Scapbtopys

coychll, an animal found In desert areas with access only

to temporary ponds and soll moisture, tolerated a loss of

45% before dehydratf onal death.

2

The phystologtcal basts for tnterspeclf lc dehydration

tolerance has recently begun to emerge. A terrestrlal

vertebrate in negative water balance Is necessarf ly

dependent upon stored water. A positive correlation exists

between Increased capacity of anuran urinary bladders and

terrestrlal Ism. Bladder capacity ranges between 1% body

mass for X... laevLs to 44% In .a.. cognatus (Bentley 1966b).

Rulbal (1962), Shoemaker (1964), and McClanahan (1967) have

shown that bufonlds and pelobatlds maintain normal body

flufd concentrations until bladder stores are depleted.

Since anurans lack salt glands and kidneys stop urine

formation during dehydration, the osmotic concentration of

body fluids must Increase after depletton of bladder water.

Shoemaker (1964) has formulated the fol I owing equation to

describe the Increase In plasma electrolytes with

dehydration:

where

Cf=Co*CBWo/CBWo-WD>

Cf=flnal plasma osmolarlty

Co=ortglnal plasma osmolarlty.

BWo= ortglnal body water content

WD=water def lclt

Further bases for graded dehydration tolerance were

found by Hf I Iman (1976, 1978). Terrestrial species

3

a.. cognatys and .S.... coychll have Increased oxygen storage

capabll lttes and maximum oxygen consumptions CVo2 max). He

found positive correlations between ventricle mass and

Vo 2 max, a stgnlf lcant decrease In Vo2 max with

dehydration, and an Increase In whole animal lactate at

critical activity point Closs of righting response). The

lmpl tcatlon Is that terrestrial Ism Is associated with

selection for Increased aerobic capacity and that

dehydratlonal death In the species Investigated Is brought

on by a reduction In circulatory oxygen del Ivery.

Poss Ible mechanisms for reduct ton In o2 del tvered to

the tissues Include the effects of hyperosmolal tty,

hypovolemta, and viscosity. Investigations of the

contractile performance of cardiac muscle bathed In

hyperosmottc solutions have shown both postlve <Koch-Weser

1963; Atkins et al. 1973), and negative lnotroptc effects,

CWtldenthal 1975; Htl Iman 1984). Ht II man (1978, 1980)

demonstrated Increased tolerance for plasma sodium tn

terrestrial species and suggested that a necessary

adaptation for extending deydratlon tolerance Is to extend

the Intrinsic osmotic I lmlt. This effect of electrolytes

was found to be Independent of hematocrlt and thus

viscosity effects.

Compensation associated with hypovolemtc stress was

shown by Hf I Iman and Sommerfeldt (1981) to Include a

redistribution of blood flow to the head, enhanced return

of lymph to the plasma space, CHll Iman and Zygmunt,

unpubl I shed), and an Increase of resting heart rate

CHll Iman 1978). In this manner brain function and cardiac

output Is maintained.

Blood flow rate Is Inversely proporttonal to blood

viscosity. During dehydration, blood viscosity Increases

after bladder and lymph flutd volumes have been exhausted.

Any compensation In ctrculatory resistance tnvolvtng

dtlatton of pertpheral vessels ts counter-productive since

the appropriate response to hypovolemta ts constriction of

the perlpheral vessels.

4

To summarize, dehydrattonal death In anurans appears

to be the result of curtatlment of oxygen del Ivery to the

tissues, resulttng from hypovolemta, hypervtscostty, and

hyperosmolal lty. Consequently, adaptations which fact I ltate

oxygen del Ivery with dehydration should enhance tolerance.

The objectives of this study were to Investigate aspects of

the effects of Increased plasma electrolytes during

dehydration on RBC function In .B.... catesbelana and

~mart nus.

Two aspects of red eel I function under hyperosmottc

stress are germane to the question of oxygen del Ivery. The

first Is wheth~r the red blood eel Is volume regulate or not

with hyperosmolal lty. Cel I deformabtl tty Is a functlonal

consequence of eel I volume. Blood viscosity Is related to

eel I deformabll tty, hence eel I volume. Mechanisms which

5

reduce blood viscosity with dehydration wtl I enhance oxygen

del Ivery and be adaptive relattve to dehydrattonal

tolerance. The second question ts whether the

hyperosmolal tty Influences the oxygen loadtng

characteristics of the blood. Ionic Interactions are known

to mediate hemoglobln oxygen loadlng. During dehydration

tntracel lular tonic strength Increases, therefore the

effects of hyperosmolal tty on oxygen dissociation curves ts

central to understanding hyperosmottc stress on oxygen

del Ivery.

One aspect of the lnabll tty to regulate plasma

electrolytes ts that eel Is wll I be bathed In hyperosmotlc

flutds. Net movement of water Is dependent upon

extracellular osmolal tty. In hypertontc soluttons, eel I ·

volumes wll I decrease unless tntracel lular osmolyte

concentrations are Increased to match extracellular

osmolal tty. A number of Investigators have examined volume

regulatton of eel Is In anlsotonlc media. Shoemaker (1964)

found retention of water f n skeletal muscle, I Iver, kidney,

lung, and heart In the toad .a.... marlnus after dehydration to

80% of orlglnal mass. He associated this volume regulatlon

with an Increase In eel lular electrolytes, CNa+, K+, Cl-).

Katz (1978) found dlfferentlal volume regulatlon In tissues

of .a.... ylrldls. A reduction In water content between 15-30%

was found for erythrocytes, muscle, and I Iver. Heart water

content changed very I lttle after adaptation to 500 mOsm

6

NaCl. Oubaln C10-3M>, an active transport blocker,

affected tonic composition, but not eel lular water content.

Studies of erythrocytes In hypotontc media have shown

volume regulation to be the result of either reduction In

eel I free amino acids CFugel I I 1967; Costa and Pierce

1983), or eel I electrolytes CKregenow 1971a). Duck red

eel Is In hypertonlc media Increase eel lular Na+ and K+

content after an lnltlal period of eel I shrinking CKregenow

1971b).

Red eel Is In anlsotonlc solutlons change surface to

volume ratio and packing of eel I hemoglobin. These factors

potent I ally affect blood viscosity. Investigators have

reported confl feting results. Melselman et al. (1967)

found hypertonlc suspensions of red eel Is to have Increased

viscosity, whereas eel Is In hypotontc solutton had reduced

viscosity relative to controls. Rand and Burton (1964)

found red eel I membranes to be more easily deformed In

hypertonlc solutions. Increased deformabtl lty should yleld

a reduced viscosity <Braasch 1971). I have Investigated·

blood viscosity over a range of hematocrfts In salt loaded

red blood eel Is. These experiments answer questions

regarding posslble viscosity advantages ln regulating vs

nonregulattng nucleated red eel Is. A reduction In viscosity

offers an obvious advantage In oxygen del.lvery.

If erythrocytes maintain volume as a consequence of

intracellular uptake of electrolytes, or if they simply

7

lose water, It would be expected that the oxygen

dissociation curve would be shifted to the right, due to an

Increase In lntracel lular Ion concentration C Rossl-Fanel I I

et al. 1961; Brunorl et al. 1975). A rightward shift of

the curve would be a useful adaptation for promoting the

oxygen supply to the tissues by decreasing hemoglobin

affinity for oxygen Clenfant et al. 1970; Metcalfe and

Dhtndsa 1970). Fact I ttatton of o2 del Ivery at the tissues

would offset general clrculatory Insufficiency which

accompanies dehydration. I have undertaken experiments to

determine the half saturation point of hemoglobin In

control and dehydrated animals. The half saturation point

of hemoglobin, CP 50>, Is a measure of the position of the

oxygen dtssoctatton curve.

·In summary, the thrust of this study ts to elucidate

factors for possible compensation of problems Involved with

q 2 del Ivery durl ng dehydration In anurans.

These factors arise from reduced eel I volume In hypertonlc

media, or from Increased tntracel lular tonic concentration

with dehydration. A comparison between species of

differing tolerance to dehydration, Cau.tg>.Ba.n.a>, may

Identify adaptations for terrestrial radiation In anurans.

8

MaterJals and Methods

An Ima ls

.BJLtg marlnys (mean mass=253g) and .B.A.n.A catesbelana

(mean mass•438g) were purchased from commercial suppl lers.

B... maclnys were maintained In the lab between 17-20oC on a

sand substrate. Pans of dlstf I led water were Included

within the enclosure for rehydration •

.B... catesbelana were kept In a sheet metal enclosure with

avaf lable water and were used within two weeks of arrival

In the lab.

Mean Cel I Volume

Mean eel I volume Cmcv> determinations were made using

a Coulter Counter, (model Zbl), and Channel lzer, (model

C-1000), Interfaced with a Tektronix 4051 computer. The

counter produces a pulse which Is In prf nclple proportfonal

to the volume of suspensfon electrolyte dfsplaced by a eel I

wlthfn the countf ng aperture. The current pulse Is

theoretically assumed to be caused by an fnsulatlng

particle movf ng within a conducting medium, and this

predicts that mcv measurements wll I be affected by eel I

membrane charge. Adams and Gregg (1972) found that errors

f ntroduced by eel I charges are lnslgnlf lcant due to the

9

comparatively large resistive current of the electrolyte.

Additional errors due to eel I path, tumbl Ing of the eel I,

and adherent eel Is passing through the aperture as

doublets, were reduced by use of an electronic editor on

the model C-1000 Channel lzer. Coulter Electronics suggest

eel I counts be less than 40,000 eel ls/ml for the 100 micron

aperture tube to reduce counting errors, a procedure always

fol lowed In these experiments. Further correction for the

above anomal Jes was made by using human red blood eel Is In

Isotonic solutlon to cal lbrate the counter and channel lzer.

Since experiments required eel Is to be suspended In

electrolyte of Increasing tonlclty, mcv determinations for

a 23.2 micron diameter latex bead (Coulter Electronics)

were made In salt solutions covering the range experienced

by dehydrated individuals. For sodium concentrations

between 100-250 mM, mcv as determined on the Coulter

counter was within 1% of Its calculated volume assuming the

latex particle to be a sphere.

Volume regulation experiments fel I Into two

catagorles. Red blood eel I Crbc) volume was fol lowed

during dehydration In .B.a. marlnus Cn=6) and .B... catesbelana

(n=6). Animals were weighed after their bladder was

drained by cloacal cannulatlon. After control blood

samples were obtained by ventrlcular puncture, anlmals were

placed In screened piastre cages and subsequently lost 6-9%

of Initial body mass/day. Dally blood sampl Ing continued

1 0

untll water losses of 35% lnltial body mass In .BJU.g and 24%

tn .B.A.D.A were achieved. Control and al I subsequent blood

sampl Ing was made by ventricle puncture. Blood was

collected In heparlnlzed tubes and a quantity of whole

blood was centrifuged at 4,000 rpm for 2 min. Hematocrlt

was recorded and plasma sodium and potassium determined on

an IL model 143 flame photometer. Red eel Is which were to

be analysed by the Coulter counter were suspended In

Ringer's Isotonic for the nonpermeant sodium ton, and

containing 5 mM KHC0 3, 1 mM CaCl2

, 5 mM glucose.

Addttlonal experiments were performed to describe the

effects of temperature, extracellular potassium, and the

function of the Na+/K+ pump on eel I volume regulation.

Blood obtained- by heart puncture from hydrated .fL. marlnus

and B.... catesbelana was suspended In Ringer's soluttons

ranging from Isotonic to soluttons containing 225 mM Na+.

These suspensions were maintained at either 20°c or 9°C and

mcv determinations made every 15 minutes for 2 hours. A

comparison of the degree and time course of volume

regulatton was made between eel Is maintained at the two

temperatures.

In order to Investigate posstble tonic contrfbuttons

to volume regulation, red eel Is from hydrated a.. martnus

and B.... catesbelana were placed In hypertontc media which

were either potassium free or contained oubain at a

concentration of 10-3M. At this concentration, oubaln

+ + blocks the operation of the Na /K pump CKregenow 1971b).

Mean eel I volume comparisons were made between eel Is In

+ hypertontc normal, K free, and oubatn media.

Hemoglobin Oxygen Affinity

Hydrated .B.a. marl nus Cn=12) and .IL. catesbelana

1 1

Cn=12) were doubly pithed, after which the ventricle was

exposed. A 4 ml sample of blood wa~ collected In a syringe

containing heparin. Blood from dehydrated .BJll.Q (35% loss

lnttlal body mass, n=9) and Rall (20% loss lnltlal body

mass, n=6) was slml I art ly collected.

Measurement of the half saturation point of

hemoglobln CP50 > was made using the technique of Edwards

and Martin, (1966). Four ml of blood was divided between 2

test tubes and each tube sealed by rubber stopper. Two 18

gauge needles on which 3-way stopcocks had been mounted

al lowed ff I I Ing of each test tube with gas mixtures under a

positive pressure. Within these equtl tbratlon tubes, whole

b I ood was f u I I y oxygenated .(gas mt xture 20% 02 , 5% C02

,

balance N2 > or fully deoxygenated Cgas mixture 5% C02 ,

b a I a n c e N2 ) • A f t er a n e q u I I I b r at I on of 4 5 m I n , 1 5 0 u I

oxygenated blood was Injected In a 300 ul capll lary tube,

fol lowed by Immediate Injection of 150 ul deoxygenated

blood Into the column of saturated blood. This volume of

blood was mixed anaerobtcal ly and analyzed for Po 2, pH, and

Pco 2 at 19°C using an IL model 313 blood gas analyzer.

12

Since equll lbrtum oxyhemoglobtn saturation Is dependent

upon the relatlve volume contribution of saturated Hb to

the total volume of mixed blood, the Po 2 of a mixture of

equal volumes of oxygenated and deoxygenated blood Is the

P50· Determinations were made at a temperature of 19°c, pH

7.55, and Pco2 of 40 mmHg.

lntracel lular Sod tum and Potassium

Approximately 2 ml of blood was collected by heart

puncture from fully hydrated .B... marl nus Cn=9) and

.B... catesbelana Cn=7). lndtvtduals were subsequently

dehydrated untll they lost 35% of Initial body mass In .BJL.tg

and 25% Jn .B.a.n.A. Animals were doubly pithed and the

ventrlcle exposed for blood col lectlon In heparlnlzed

syringes.

Blood was centrifuged at 4,000 rpm for 10 min In

300 ul caplllary tubes. Plasma and white eel I fraction

were removed and the red eel Is were blown Into test tubes

weighed to the nearest 0.1 mg, (Sauter model 414 balance).

Red eel Is were dried to constant mass at 40°c. Two ml of

0.8N HN03 was used to extract Na+ and K+ from the eel Is.

Al lquots of 100 ul, 500 ul, and ml were evaporated to

dryness In order to concentrate tons. Lithium dlluent was

added and determinations made on an IL model 143 flame

photometer. After correction for trapped plasma, C10% wet

mass), lntracel lular concentrations were expressed as

meQ/Kg dry mass eel Is.

Viscosity

au.t.g marlnys Cn=3) were doubly pithed and the

ventrlcle exposed. Blood was collected Jn test tubes

containing ammonium heparin and centrifuged at 4,000 rpm

for 5 min. Separated plasma was salt loaded to a

concentration of 180 meQ NaCl. Red eel Is were then

suspended In salt loaded plasma In order to manufacture

hematocrtts between 0-77%. A 200 ul sample of suspended

eel Is was lmmedlately analysed at 2ooc with a

Wei ls-Brookf teld cone/plate viscometer model LVTDCP.

Viscosity reported was for a shear rate of 450/sec. RBC's

In hypertontc media tnftfal ly behave as osmometers before

gaining water <Figure 2). Cel Is placed In salt loaded

plasma for Immediate viscosity determinations are thus

termed nonregulatlng. Comparison was made with viscosity

of red eel Is In Isotonic plasma of hydrated au.t.g <Hedrick,

unpub I I shed).

RESULTS

Mean Cel I Volume

The results of In vivo regulation of red eel I volume

In .B... catesbelana and~ marlnys are shown In Figure 1.

13

Mean eel I volume of hydrated .B... catesbelana Cn=4) was 770 ±

1 4

20 u3 <±se). Loss of 14-20% of lnltlal body mass resulted

In a mcv of 771 + 18 u3. Mean eel I volume of a._ marl nus

Cn=4) red eel Is was 460 ± 11 u3, and 451 ± 11 u3 after

water losses ranging from 28-36% of lnltlal body mass.

These data Indicate volume regulation since predicted

reduction of Tnltlal volumes should equal 15% In Ra.

catesbelana and 28% in lL. marl nus if these eel Is acted as

perfect osmometers in hypertonic plasma.

The mechanism of volume regulation is temperature

dependent as shown in Figure 2 for both species. Recovery

of eel I volume after challenge by hypertonic medium, (180

mEq Na+), occurs within 45 minutes at 2ooc, whereas at goc,

recovery Ts incomplete after 2.5 hours.

The results of In vitro experiments involving

regulation In potassium free solutions and normal Ringer's

containing oubaln c10-3 M> ls shown for Ra. catesbeiana

(Figure 3) and lL. marinus (Figure 4). The relationships

between percent decrease of Initial eel I volume and media

toniclty were similar in both species. Cel Is placed in K+

free media behaved as simple osmometers and did not volume

regulate Cp>0.05). In a similar fashion, slopes of

regression I Ines for eel Is in normal and oubaln Ringer's

were not different Cp>0.05). A significant decrease in

slopes -was found between predicted osmometer and eel Is In

normal Ringer's Cp<0.01>, or eel Is in Ringer's plus oubain.

Evidently, extracellular potassium Is required for the

observed volume regulation, but otibaln has no effect.

Hemoglobin Oxygen Aff lnlty

1 5

No difference Cp>0.05) was found between hydrated and

dehydrated hemoglobin oxygen affinities In either species.

P5 0 was deter m l n e d to be 4 8 • 8 ± 1 • 4 torr C n = 1 2 > a n d 5 0 • 6

± 1 .6 torr Cn=6), respectively, tn hydrated and dehydrated

.B.a. catesbelana. Oxygen aff lnlty was greater tn .a.... marlnus;

41 .6 ± 1.4 torr Cn=12) In hydrated animals and 43.0 ± 0.5

torr Cn=9) tn dehydrated Individuals.

lntracel lular Sodium and Potassium

The relationships between plasma sodium and

tntracel lular ton concentration were similar for both

species <Figures 5 and 6). A significant Increase of

lntracel lular K+ and Na+ concentrations accompanied

dehydration In both species Cp<0.05). Results are I lsted

In Table 1.

Viscosity

Figure 7 shows the viscosity relatlonshlps for

.a.... marl nus red eel Is In Isotonic and hypertonlc (180 mEq

Na+) solutions. The slope ts significantly lower Cp<0.005)

and the Intercept higher Cp<0.001) In salt stressed eel Is.

A higher Intercept may be the result of salt Interactions

with plasma proteins. Control plasma viscosity (1.8 ±

0.04cP) was lower than viscosity of plasma loaded to 180

mEq Na+ C2.6 ± 0.07cP>.

16

TABLE I

INTRACELLULAR SODIUM AND POTASSIUM CONCENTRATIONS

CMEQ/KG DRY MASS) IN THE ERYTHROCYTES OF

.6.&, MARINUS CN=9) AND B--. CATESBEIANA CN=9).

VALUES ARE MEANS ± STANDARD ERROR ASSUMING

10% TRAPPED PLASMA.

ASTERISK INDICATES SIGNIFICANT DIFFERENCE CP<0.025).

SPECIES CONTROL

.6.&, marl nus Na+ 40.4 ± 1 .7

K+ 234.8 ± 2.3

B--. catesbelana Na+ 34.1 ± 2.3

K+ 229.0 ± 3.4

DEHYDRATED

73.2 ± 8.7*

260.6 ± 6.0*

58.3 ± 3.7*

269.o ± a.a*

17

825

775

450

400 100

1 8

RANA

BUFO

140 180 220

PLASMA SODIUM (mEq/L)

Figure 1. In vivo volume of erythrocytes of .a.a, marlnus

(water losses 28-36% lnltlal body mass)

and .B... catesbelana (water losses 14-20% tnltlal

body mass).

800 RANA

750 •

700 ......... C')

:a. ......... rzl 650 ~ ::J ~ BUFO 0 > 800 ~ ~ 400 rzl 0 Q r:l ~

~ < r:l ~

350

300 0 80 120

TIME IN MINUTES

Figure 2. Temperature· dependence of RBC volume

regulatlon In .IL. marlnus and .B.a. catesbelana. Rana,

<•9°C Isotonic,• 20°c lsotonrc,&9°C hypertonrc,

e20°c hyperton r c). .EW.f.g, (. 9°C r soton I c,

e20°c lsotonlc~9°C hypertonlc~20°c hypertonlc).

1 9

ra1 :a = ..:I 0 .> ..:I < .... ~ .... :z: ..... pa Cl2 < ~ = 0 ~ Q ~

40

30

20

10

o.o 1.0 a.a o.e . .

CQAJll2l.t fl.ASMA Na · AL MEDIA Na

0.4

Figure 3. Volume regulation of RBC's In .B.a.

catesbe I ana after 1 .5 hours. Top I I ne Ind I cates

behavior as osmometer, y=71.5 <±0.4)x-0.1 <±0.1),

r=1.00. Bottom I lne RBC 1 s In normal Ringer's,

y = 2 1 • 0 C ±5 • 3 ) x - 0 • 4 ( ±2 • 0 ) , r = • 7 8 •

CA , potassium free media; Y , oubaln media;

•, normal Ringer's)

20

~ ::a = ~ 0 ·> ~ < .... e.. .... :z ..... Pa Cll < ~ g:: c ~

·Q . 'if.

3

20

.10

1.0 o.a o.e.

CONTROL PLASMA Na FINAL MEDIA Na

21

0.4

Figure 4. Volume regulatton of RBC's Jn .6.a. martnus

after 1 .5 hours. Top I lne ts behavior as osmometer,

y=66.7 <±0.5)x+0.1 <±0.1), r=1.00. Bottom ltne

RBC's In normal Ringer's, y=26.9 <±2.B>x-0.3 <±1.1),

r=.94. C~, potassium free media; 9 , ouba f n

media; ., normal Ringer's)

_..... .a 280 ·~

~ .... "tS

bl ... ' ct Ci:l 240 a -aJ z 0 ...... ~ < 200 ~ :.:::> ~ ~

80 rzl 0 • < ~ E-t z .....

40

•

0

100 140 180

PLASMA SODIUM (mEq/L)

Figure 5. The relatlonshtp between red eel I

lntracel lular ton concentrations and plasma

electrolytes In~ catesbelana corrected for 10%

trapped plasma.

(~sodium y=O.Sx-21, r=.75; •potassium y=1.1x+97,

r=.93).

22

23

-- • +a 280 • • ~ ~ ... .,, bf)

::id ' : 240 s ~

a.l z 0

. 1-4

~ 200 < • •

~ ::::> • ~ ...:i r.J:l 0 80 < ~ E-4 z 1-4

40 •

0

100 140 180

PLASMA SODIUM (mEq/L)

Figure 6. The relatlonshlp between red eel I

lntracel lular Ion concentrations and plasma

electrolytes In .£L.. marlnus corrected for 10% trapped

plasma.

ce sodium y=0.5x-14, r=~73; ~potassium y=0.4x+190,

r=.79).

24

Figure 7. The relationship between .B.a. marlnus

hematocrlt and the In transformation of red eel I

viscosity at a shear rate of 450/sec and 20°c. Sol Id

I lne Is salt loaded (180 mEq/L), In y=0.028

<±.001 )x+0.90 <±.04), r=.98. Dashed I lne Is

Isotonic plasma. In y=0.035 <±.002)x+0.43 <±.07),

r=.93.

~ \ ~

\ \ .

\

~ \

\ \

\

0 0 ~ c.j ·~ ... (d:>) A..LISOOSIA '1'13:0 111

. .

\

0

0 co

0 co

0 ..

0 ~

0

25

-r/.l ...:I ...:I rzl 0 (/e. ..__

E-4 ..... ~ 0 0 E-4 < :il rzl

=

26

DISCUSSION

.B... catesbelana·and .B... marlnys regulate red blood eel I

volume during dehydration, (Figure 1). In vitro

experiments In hypertonlc media Indicate slmllar responses

for red blood eel Is of both species and a common mechanism

for volume regulatlon. Figure 2 shows the biphaslc nature

of volume regulation In RBC 1 s. lnitlal ly, eel Is In

hypertonlc media shrink, fol lowed by volume recovery within

45 min for eel Is maintained at 2ooc. The lnltlal shrinkage

Is not slgnlf lcantly different from eel I behavior as an

osmometer. Kregenow (1971b), Cala (1977), and Amende and

Pierce (1980), demonstrated a slmllar blphaslc volume

regulation for red eel Is In ducks, flounder, and mol I uses.

Shown by Figure 2 Is the temperature dependence of volume

regulatlon, since cells maintained at 90C require a longer

time to regain volume In hypertonlc media than eel Is at

2ooc. Temperature dependence Indicates a mechanism

lnvolvlng an active uptake of plasma osmolytes. Addltlonal

experiments were designed to test volume regulatlon In the

face of a Na+/K+ pump blockade by oubaln c10-3M), as wel I

as eel I osmotic behavior In potassium free Ringer's • .B...

catesbelana (Figure 3) and .a.. marlnus (Figure 4) show a

regulatory behavior In K+ free solution which Is not

27

stgntftcantly different from an osmometer Cp>0.05). Oubatn

falls to affect volume regulatton since this treatmen--t ts

not slgnlf fcantly different from eel I behavior In normal

media. These results are In agreement with data presented

for red eel Is of ducks and polychaete worms by Kregenow

C1971b) and Costa and Pierce (1983).

Figure 5 <.B.a.n.A> and Figure 6 (BJL!.s2) demonstrate

sodium and potassium uptake during dehydration, Indicating

the Importance of these cations In maintenance of RBC

volume. Schmidt-Nielsen (1975) found sodium permeabll tty

Increased for flounder red blood eel Is In hypertontc media.

Kregenow C1971b) showed an Increase of tntracel lular

potassium In duck red eel Is exposed to hyperosmottc shock.

Volume regulatlon requires extracellular K+ avallabll lty.

Potassium Is accumulated against a concentration gradlant

In both species. Schmidt and McManus (1974) describe a Na+

and K+ uptake which Is oubatn Insensitive and fact I ltates

cation movement during volume regulatlon. Nakao et al.

(1963) Isolated from human red blood eel Is a Na+, K+ ATPase

which was not Inhibited by oubaln.

To summarize, volume regulatlon of RBC's In the two

anurans studied rs achieved by active uptake of plasma

potassium via an oubaln Insensitive pump and movement of

plasma sodium down a concentration gradient. ·These results

are In agreement with previous studies. In addition, this

study demonstrates volume regulation and Ionic uptake

28

during dehydration. Previous studies have largely Involved

mammal Jan or avian eel Is which do not normally experience

markedly Increased plasma electrolytes with dehydration.

Volume regulatton achieved by uptake of Ions offers

an opportunity to offset general clrculatory lnsuff Jclency

accompantng dehydration. The effect of Ionic mediators of

hemoglobf n function Is to reduce Hb oxygen aff lnlty. The

effect of salts Jn lowering oxygen aff tnfty ts thought to

reflect preferential binding of salts by deoxygenated as

opposed to the oxygenated form of hemoglobin CTyuma 1974).

A shift of the oxygen dissociation curve to the right has

been tmpl lcated In the adaptation to anemia and hypoxic

hypoxia In sheep Clenfant et al. 1970). In this manner,

Increased tntracel lular electrolytes could substantially

Increase del Ivery of oxygen to the tissues and be adaptive

In terrestrtal species to extend tolerance to dehydration.

Control ~ 0 determinations for both species are In

agreement with publ I shed values establ tshed under similar

conditions (Hal I, 1968; Tazawa et al., 1979).

Dehydrated Individuals do not have a slgnlftcantly

different oxygen aff Jnlty. Increased lntracel lular

concentrations do not prove to be adaptive for del Ivery of

oxygen during dehydration In either species. This

seemingly anomalous finding may be explained In terms of

Increased oxygen affinity caused by other modlf ters of Hb

function such as organic phosphates which override the

effects of salts.

29

Blood flow rate Is Inversely proportlonal to blood

viscosity. Blood viscosity ts a combined term which

Includes the viscosity of the two componants of whole

blood, blood plasma and red blood eel Is. The non-Newtonian

behavior of whole blood ts associated with substanttal

protein concentrations and suspended red eel Is.

~ martnus plasma viscosity Increased with Increased plasma

sodium. Ionic Interactions with plasma protein were

presumably responstble for the slgnlf tcantly higher

Intercept for salt loaded blood CFtgure 7).

Red blood eel I viscosity ts dependent on shape,

volume, membrane rfgfdtty, and mean corpuscular hemoglobtn.

concentration CMCHC). Erslev and Atwater (1963) found

nearly a doubl Ing of viscosity as MCHC Increased from

24%-38%. In~ marl nus, the slope of the I tne tn Figure 7

for salt loaded eel Is, Cnon-regulatlng), Is slgnlftcantly

lower than the slope of the I tne for eel Is tn normal

plasma. These slopes represent eel I viscosity st nee the

second component of the slope, plasma viscosity, remains

constant. I therefore argue that nonregulattng red blood

eel Is tn hypertontc plasma are more dtstenstble and

therefore less viscous than normal eel Is In Isotonic

plasma, (dashed I fne, Figure 7). Rand and Burton (1964)

found human red eel I membranes tn hypertonfc solutfon C1.2%

30

NaCl), to be more dlstenslble than membranes In Isotonic or

hypotonlc media. Melselman et al. (1967) found human red

eel Is In hypertonlc plasma to be more viscous than eel Is In

hypotonlc plasma. Their reported values lack estimates of

varlabll lty and may merely reflect Increased plasma

viscosity.

Within a physlologlc range of hematocrlts, blood

viscosity In .a... marl nus Is lower for red blood eel Is which

regulate volume. This may not represent a true viscosity

advantage for regulatlng eel Is, since If correction Is made

for plasma viscosity, the previous advantage disappears.

Constraints for regulatlon of eel lular volume In red blood

eel Is may therefore Include factors other than viscosity.

One such selective pressure for maintenance of eel I volume

might be the proper function of membrane bound enzyme

systems.

In summary, ~ catesbelana and .a... marlnus maintain

red blood eel I volume during dehydratlonal stress by uptake

of sodium and potassium. Increased lntracel lular Ionic

concentrations do not alter oxygen del Ivery by shifting the

oxygen dissociation curve to the right. Hypertonlc plasma

does not appear to Increase red eel I viscosity although

whole blood viscosity Is higher for hematocrlts less than

70%. Increased viscosity In salt loaded blood may be

largely attributed to Increased plasma viscosity.

Volume regulatlon of red eel Is occurs but Is Insufficient

to cancel the effect of Increasing plasma viscosity.

31

REFERENCES

Adams, R.B., and Gregg, E.C. (1972) Pulse shapes from particles traversing Coulter oriface fields. Phys. Med. Biol. 17(6):830-842

Amanda, L.M., Pierce, S.K. (1980) Cellular volume regulation in salinity stressed mol I uses: The response of Noetia ponderosa CArcidae) red blood eel Is to osmotic variation. J. Comp. Physiol. 138:283-289

Atkins, J.M., Wildenthal K. and Horowitz, L.D. (1973) Cardiovascular responses to hyperosmotic mannitol in anesthetized and concious dogs. Am. J. Physiol. 225:132-137

Bentley, P.J. C1966a) Adaptations of amphibia to arid environments. Science 152:619-623

Bentley, P.J. C1966b) The physiology of the urinary bladder of amphibia. Biol. Rev. 41 :275-316

Braasch, D. (1971) Red cell deformability and capillary blood flow. Physiol. Rev. 51(4):679-700

Brunort, M., Falcioni, G., Fortuna, G., and Giardina, B. (1975) Effect of anions on the oxygen binding properties of the hemoglobin componants from trout CSalmo irideus>. Arch. Biochem. Biophys. 168:512-519

Cala, P.M. (1977) Volume regulation by flounder red blood cells In anisotonic media. J. Gen. Physiol. 69:537-552

Costa, C.J., and Pierce, S.K. (1983) Volume regulation in the red coelomocytes of Glycera dibranchiata: An interaction of amino acid and K+ effluxes. J. Comp. Physiol. 151:133-144

Edwards, M.J. and Martin, R.J. (1966) Mixing tecnique for the oxygen-hemoglobin equil tbrium and Bohr effect. J. Appl. Physiol. 21(6):1898-1902

Erslev, A.J., and Atwater, J. (1963) Effect of mean corpuscular hemoglobin concentration on viscosity. J. Lab. Cl in. Med. 62:401-406

Fugellt, K. (1967) Effect of cell volume In flounder erythrocytes accompanied by a decrease in plasma osmolartty. Comp. Biochem. Physiol.· 22:253-260

Hal I, F.G. (1966) Hemoglobin functions Jn the blood of .B.1Lf..Q marlnus. J Cel I Physiol. 68:69-73

33

HJ I Iman, S.S. (1976) Cardiovascular correlates of maximal oxygen consumption rates In anuran amphibians. J. Comp. Phystol. 109:199-207

Ht I Iman, S.S. (1978) The roles of oxygen del Ivery and electrolyte levels in the dehydratlonal death of Xenopus laeyis. J. Comp. Physiol. 128:169-175

Hi I Iman, S.S. (1980) Physiological correlates of differential dehydration tolerance in anurans. Copeia 1:125-129

Hil Iman, S.S., and Sommerfeldt, R.W. (1981) Mlcrosphere studies of amphibian systemic blood flow redistribution during dehydration, hypovolemia, and salt load. J. Exp. Zool. 218:305-308

Hi I Iman, S.S. (1984) lnotroplc influence of dehydration and hyperosmolal solutions on amphibian cardiac muscle. J. Comp. Phys. 154:325-328

Katz, U. (1978) Ionic and volume regulation in selected tissues of the euryhal ine toad .B.1Lf..Q ytrtdts. Alfred Benzon Symposium XI, Munksgaard

Kock-Weser, J. (1963) Influence of osmolarlty of perfusate on contractil tty of mammal Ian myocardium. Am. J. Phystol. 204:957-962

Kregenow, F.M. C1971a) The response of duck erythrocytes to nonhemolytic hypotonic media. Evidence for a volume-control I Ing mechanism. J. Gen Phys fol. 58:372-395

Kregenow, F.M. C1971b) The response of duck erythrocytes to hypertonlc media. J. Gen. Physiol. 58:396-412

Lenfant, C., Torrance, J.D., Woodson, R., and Finch, C.A. (1970) Adaptation to hypoxia. In .Bil~ Metabol Ism ..a.n.d. Function. ed. G.J. Brewer, Plenum Press, New York pg. 203-212

McClanahan, L. (1967) Adaptations of the spadefoot toad Scaphlopus coychll, to desert environments. Comp. Blochem. Physlol. 20:73-99

Meise Iman, H.J., Merril I, E.W., GI 1111 and, E.R., Pel letter, G.A., and Salzman, E.W. (1967) Influence of plasma osmolarlty on the rheology of human blood. J. Appl. Physlol. 22(4):772-781

34

Metcalf, J. and Dhtndsa, D.S. (1970) A comparison of the mechanisms of oxygen transport among several mamma I I an species. In fuut ~ Metabo I t sm a.ru1 Fynctlon. ed. G.J. Brewer, Plenum Press, New York pg.229-240

Nakao, T., Nagano, K., Adachi, K., Nakao, M. (1963) Separation of two adenoslne triphophatases from erythrocyte membrane. Blochem. Btophys. Res. Comm. 13:444-448

Rand, R.P., and Burton, A.C. (1964) Mechanical properties of the red eel I membrane. Btophys. J. 4:115-135

Rossl-Fanel I I, A., Antonini, E., and Caputo, A. (1961) Studies on the relations between molecular and functional properties of hemoglobin. J. Btol. Chem. 236(2):397-400

Rulbal, R. (1962) The adaptive value of bladder water In the toad, .B.u..f..Q cognatys. Phystol. Zool. 35:218-223

Schmldt,W.F., and Mcmanus, T.J. (1974) Furosemtde­sensltive co-transport of Na+ plus K+ Into duck red eel Is activated by hypertonlctty or catecholamlnes. Fed. Proc. 33(5):1457

Schmidt-Nielsen, B. (1975) Comparative physiology of cellular ton and volume regulation. J. Exp. Zool. 194:207-220

Shoemaker, V.H. (1964) The effects of dehydration on electrolyte concentrations in a toad .B.u..f..Q marlnys. Comp. Blochem. Physlol. 13:261-271

Tazawa, H., Mochizuki, M., and Pllper, J. (1979) Blood oxygen dissociation curve of the frogs .B.a.n.A catesbelana and .B.a.n.A breyipoda. J. Comp. Physlol. 129:111-114

Thorson, T.B. (1955) The relatlonshlp of water economy to terrestriallsm In amphibians. Ecology 36:100-116

Tyuma, I. C 1974) Gas transport. In Ce! lu!ar ~ Molecular Bio logy j2.f Erythrocytes. ed. H. Yoshikawa and S.M. Rapoport University Park Press, Baltimore pg.279-305

Wl!denthal, K., Adcock, R.C., Crle, J.S.,

35

Templeton, G. H (1975) Negative lnotroplc Influence of hyper-osmotic solutlon on cardiac muscle. Am • J • Phy s I o I • 1 5 0 5 - 1 5 0 9